Mounting

techniques
 Whole mounts

• There are many specimens that are most satisfactorily mounted

and observed as a whole. Though whole mounts do not require
sectioning, some amount of trimming of excess tissue may be
required.
• Filamentous algae, mycelia of fungi, prothalli of ferns, epidermal
peelings, small and transparent gametothalli of liverworts, etc. are
best mounted as such in a suitable mounting medium on a clean
plain microslide, covered with coverslip and used for microscopic
observation. Such micropreparations are called whole mounts.
• Smears of pollen grains and spores are in reality whole mounts,
but are treated under a separate heading in that name. Whole
mounts can be temporary, semi-permanent or permanent
depending on the purpose and length of use. Whatever the type of
whole mount, the microslide and coverslip should be clean and
dry
 Temporary whole mounts

• Temporary whole mounts are prepared for routine

temporary and preliminary observation mostly for
classwork purpose. The specimen is mounted in a
few drops of water on a clean plain microslide and
covered with a coverslip.
 Procedure
• Place just enough drops of water in the central point of the
microslide.
• Transfer the specimen to the water drop.
• Take care not to trap any air bubble in the specimen.
• Hold the clean coverslip gently and lower it horizontally over the
water drop until the centre of the coverslip touches the water drop.
• Release the coverslip so that the mounting medium (water) gently
spreads reaching the edges of the coverslip.
• Some have a tendency to bring the edge of the coverslip into
contact with the edge of the water drop while supporting the
coverslip with a dissecting needle. By drawing the needle slowly,
the mounting medium is drawn to the other edge. This method has
the disadvantage of drawing the specimen also to the edge of the
coverslip. Therefore, this method is not preferred by many.
In order to mount the specimen exactly in the
centre of the slide, the following steps are to be
followed.
• Draw the outline of a microslide on a white
hardboard (the blank reverse side of printed
invitation cards should serve this purpose).
• Draw 1" × 1" outline for the label preferably on the
left side.
• Draw diagonals connecting the four corners in the
remaining space.
• Place the clean plain microslide on the outline drawn.
• The specimen is to be mounted in the point where the
diagonal lines meet (in the outline already drawn on
the hardboard).
 Semi-permanent whole
mounts.
• Whole mounts that require keeping for a little longer (a few
hours to a fortnight) are mounted in glycerine medium.
Pure glycerine or aqueous dilutions of glycerine or some
form of glycerine jelly are used for making semi-permanent
whole mounts.
• Glycerine preserves the natural colours of the plant
specimens. It has been found to be an ideal mounting
medium for semi-permanent preparations of unicellular and
colonial Chlorophyta, protonemata of mosses, fungal
spores, prothalli of ferns, etc.
 Procedure
• Place delicate specimens that are likely to plasmolyse
easily in one or two drops of 10% glycerine (aqueous)
on a clean plain microslide.
• Keep the slide undisturbed, covered under a petri dish.
• This allows the glycerine to concentrate.
• When the mounting medium has concentrated
sufficiently, place a clean coverslip over the drop of
glycerine cautiously taking care that glycerine does not
ooze out of the coverslip.
• The edge of the coverslip is sealed with a neutral nail
polish or any other inexpensive resin.
• Kill and fix the specimen.
• Stain the specimen in some aqueous stain. Only aqueous stain solutions
are to be used for mounting in glycerine jelly.

• Iron haematoxylin with or without counterstain has been found to be

suitable.
• Wash off the excess stain from the specimen before mounting.
• Place the stained specimen in the 10% aqueous glycerine in a small dish
that offers as much evaporating surface as possible.
• Leave the dish covered and undisturbed. Keeping the dish on the upper
shelf or cooler part of a thermostat may help in evaporation.
• Caution Rapid evaporation may lead to shrinkage.
• Allow enough time for the 10% glycerine to reach the consistency of pure
glycerine. Now the specimen is ready for mounting.
• Using a section lifter, take enough of the specimen and
remove excess of glycerine from the specimen using a
blotting paper.
• Place the specimen in one or two drops of warmed jelly,
just enough to spread to the edge of the coverslip. If the
jelly mixture oozes out of the edges of coverslip, it is
impossible to seal the same.
• Apply a clean coverslip, preferably a circular one.
• Sealing of the coverslip can be performed either at once
or after a week or ten days when the jelly has solidified.
• Gold-size or Canada balsam is generally used for
sealing.
• Sealing may be done free hand or using a turn table.
• Preparation of Kaiser’s glycerine jelly.
o Pure gelatin - 1 part by weight
o Water - 6 parts by weight
• Allow the gelatin to soak for at least 2 hours.
• Add glycerine 7 parts by weight.
• Add phenol crystals 1 g for every 100 g of the mixture.
• Warm for about 15 minutes, stirring constantly and vigorously
until all the flakes produced by phenol have disappeared
completely.
• Filter through cotton while still warm.
• Store the mixture in a small bottle that can be conveniently
warmed in a hot-water bath.
• Melt the jelly every time in hot-water bath prior to mounting.
• It may be required to make whole mounts of some
specimens as permanent preparations.
• The following two methods are the most commonly
used of the several methods available.
• This is the simplest method that can be attempted by even the beginners.
It has been found to work well even with difficult specimens such as
Spirogyra. It is important that each step be carried out in its logical
sequence to obtain the desired result.
• Kill and fix the specimen in a weak or medium chrome–acetic fixative
which has been found to give very good staining effects especially the
filamentous members of Chlorophyceae. FAA works out well for all
kinds of materials except aquatic forms.
• After fixation, place the specimen in a deep solid watch glass or low
stender or petri dish and carry out all the further steps in it by pouring
out the fluid every time and not transferring the specimen between
containers.
• Wash out the fixative thoroughly with running water to wash off CA
fixative and with distilled water to wash off FAA.
• Stain with an aqueous stain. Harris’ haematoxylin, iron haematoxylin
and Mayer’s carmalum are recommended.
• Differentiate the dye sharply. Take care not to
destain too much since the further steps may
remove some stain.
• Wash out the differentiating agent thoroughly.
• Dehydrate in 15%, 30%, 50% and 70% ethyl
alcohol allowing at least 20 minutes in each
concentration. Keep the container covered between
changes of fluids to avoid evaporation.
• Leave the specimen in 85% alcohol for at least 18
hours. The long immersion in 85% alcohol hardens the
tissue without making it brittle. This is very important
for the further handling of the specimen. Shortening of
this step will result in the specimen becoming
excessively brittle upon the addition of a stronger
alcohol rendering it non-amenable to further treatment.
• Counterstain with any desired cytoplasmic stain
dissolved in equal parts of 95% alcohol and methyl
cellosolve. As chances for overstaining are least, the
counterstain used may be fairly strong. About 15
minutes should be enough for counterstaining.
However, make sure that the stain has penetrated
deeply.
• Table will help in choosing the right counterstain

Primary stain Counterstain Specimen

Haematoxylin Erythrosin B Orange Rhodophyta

G Phaeophyta
Fast green Chlorophyta

Carmine Not required For most specimens

Carmine Aniline blue or Leaves or epidermal

fast green peels
• Differentiation of the counterstain is not required since the
hygrobutol to be used in the next step prevents the 95% alcohol from
extracting too much of the stain.
• Pour out the counterstain and immediately wash out the excess stain
with 95% alcohol.
• Give a change in 95% alcohol.
• Start adding hygrobutol by gradual substitution method. (Add a
small amount of hygrobutol every minute or so to avoid strong
diffusion currents. Every time mix the fluids by gently tilting the
container back and forth. Discard some of the mixture between
every third or fourth addition. Continue until the mixture has
reached an approximate proportion of 90 parts hygrobutol and 10
parts ethyl alcohol. It is not essential to remove alcohol completely.)
• Pour out most of the butyl and ethyl alcohols replacing at once with
thick balsam diluted at least ten times with hygrobutol. The
specimen should be immersed in butyl balsam of volume ten times
its own.
• Keep the container aside in a dust-free warm place.
Allow enough time until much of the solvent butanol
has evaporated leaving the balsam to a consistency
suitable for mounting.

• Take care to add enough diluted balsam so that the

specimen will not be exposed to air on evaporation.
• The process of evaporation should be gradual.
Therefore do not induce rapid evaporation by any
artificial means. The safest minimum period for
evaporation is said to be 2 hours.
• Place a fairly large drop of balsam about the size of
two grains of wheat in the centre of a clean
microslide.
• This is another equally simple and inexpensive method of
making permanent whole mounts.

The procedure is as follows:

• Kill and fix the specimen as described in the previous
method.
• Wash off the fixative thoroughly.
• Stain with an aqueous basic stain, e.g. any haematoxylin
solution.
• Transfer the stained specimen in enough volume of 10%
aqueous glycerine taken in a small container such as a
stender dish or petri dish.
• Keep the container undisturbed in a dust-free
corner.
• If at all the dish is to be covered to eliminate dust
from settling, take care to leave airspace between
the container and the lid/cover.
• Leave the set-up for a few days until the diluted
glycerine attains the consistency of pure glycerine.
• If required, the container may be kept in a hot-air
oven to accelerate the process of evaporation
• Remove the glycerine with several changes of 95%
alcohol. If the specimen is in the form of thicker
masses, it requires more number of changes.
• It is always better to place the specimen in a wire strainer
which is placed in the dish with about 1 cm of clear space left
below the strainer. This helps the heavier glycerine to settle
down free from the specimen.

• If desired, the specimen in 95% alcohol may be counterstained

with any suitable dye dissolved in 95% alcohol.
• To complete the process of dehydration, replace 95% alcohol
with absolute alcohol.
• De-alcoholize the specimen by gradual replacement method
(alcohol: xylol = 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and
finally pure xylol). Allow 5 to 10 minutes in each mixture.
• Hygrobutol may also be used in the place of xylol as the
specimen may not be so much hardened as with xylol.
• Replace xylol (or hygrobutol as the case may be)
with balsam highly diluted with xylol or
hygrobutol.
• Leave the dish undisturbed until the balsam
evaporates to mounting consistency.
• Mount the specimen in the same way described
earlier.
• It may be necessary to manipulate the specimen
transferred to the mounting medium. The specimens that
are mounted singly, such as sections, prothalli, etc., can be
manipulated with ease. Using fine scissors cut the mass of
filamentous specimens into smaller bunches of 5 mm and
less. It will be easier to mount the bunch and the
individual filaments can then be separated and spread
easily.
• Leave the slide on a warming plate for a day or two to
solidify the balsam.
• Place a clean coverslip over the drop of balsam carefully
and gently press on the coverslip with the handle of a
dissecting needle which may help in spreading the balsam
towards the edge of the coverslip. It also helps to flatten
the specimen if it is slightly curved.